Calcium-dependent cysteine reactivities in the neuronal calcium sensor guanylate cyclase-activating protein 1.

Guanylate cyclase-activating protein 1 (GCAP-1) is a Ca(2+)-sensing protein in vertebrate photoreceptor cells. It activates a membrane-bound guanylate cyclase. Three of four cysteines present in wild-type GCAP-1 were accessible to the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) in the presence of Ca(2+). Only Cys106 became exposed to the solvent after Ca(2+)-chelation. Since Cys106 is located in EF-hand 3, we could determine an apparent K(D) of 2.9 microM for Ca(2+) binding to this site with a fast off-rate (t approximately 2 ms). We conclude that the rapid dissociation of Ca(2+) from EF-hand 3 in GCAP-1 triggers activation of guanylate cyclase in rod cells.     

The nitrogen budget of a pine forest under free air CO2 enrichment

Elevated concentrations of atmospheric CO₂ increase plant biomass, net primary production (NPP) and plant demand for nitrogen (N). The demand for N set by rapid plant growth under elevated CO₂ could be met by increasing soil N availability or by greater efficiency of N uptake. Alternatively, plants could increase their nitrogen-use efficiency (NUE), thereby maintaining high rates of growth and NPP in the face of nutrient limitation. We quantified dry matter and N budgets for a young pine forest exposed to 4 years of elevated CO₂ using free-air CO₂ enrichment technology. We addressed three questions: Does elevated CO₂ increase forest NPP and the demand for N by vegetation? Is demand for N met by greater uptake from soils, a shift in the distribution of N between plants, microbes, and soils, or increases in NUE under elevated CO₂? Will soil N availability constrain the NPP response of this forest as CO₂ fumigation continues? A step-function increase in atmospheric CO₂ significantly increased NPP during the first 4 years of this study. Significant increases in NUE under elevated CO₂ modulated the average annual requirement for N by vegetation in the first and third growing seasons under elevated CO₂; the average stimulation of NPP in these years was 21% whereas the average annual stimulation of the N requirement was only 6%. In the second and fourth growing seasons, increases in NPP increased the annual requirement for N by 27-33%. Increases in the annual requirement for N were largely met by increases in N uptake from soils. Retranslocation of nutrients prior to senescence played only a minor role in supplying the additional N required by trees growing under elevated CO₂. NPP was highly correlated with between-plot variation in the annual rate of net N mineralization and CO₂ treatment. This demonstrates that NPP is co-limited by C availability, as CO₂ from the atmosphere, and N availability from soils. There is no evidence that soil N mineralization rates have increased under elevated CO₂. The correlation between NPP and N mineralization rates and the increase in the annual requirement for N in certain years imply that soil N availability may control the long-term productivity response of this ecosystem to elevated CO₂. Although we have no evidence suggesting that NPP is declining in response to >4 years of CO₂ fumigation, if the annual requirement of N continues to be stimulated by elevated CO₂, we predict that the productivity response of this forest ecosystem will decline over time.     

Les génotypes responsables de mucoviscidose ou d’absence bilatérale des canaux déférents ABCD

Over the last decade, the genetic basis for CBAVD has been identified by its association with CFTR gene mutations, and CBAVD is now generally considered to be a mild or incomplete form of CF. In this review, the author summarizes the main results of compilation of CFTR gene analysis conducted in French laboratories for 3,923 patients with CF and 800 males with CABVD. The degree of clinical expression can be affected by several variables, including the molecular mechanisms by which the various CFTR mutations impair or disrupt the function of the CFTR chloride channel. Phenotypic expression of CFTR mutational genotypes varies from severe, progressive pulmonary disease with pancreatic insufficiency (CF-PI), to mild pulmonary disease with pancreatic sufficiency (PS) or singleorgan forms of “CFTR-opathies”. In CF, a total of 310 different CFTR mutations accounting for 94% of 7,846 CF alleles have generated almost 500 different genotypes, comprising 2 severe mutations in 88% of cases (CF-PI), one severe mutation in trans to a mild mutation in 11% (CF-PS), and 2 mild mutations in 1% of identified genotypes. In CBAVD, 137 mutations scattered over the whole gene were identified in 60% of 1,600 CBAVD alleles during the study. Among the 150 characterized mutational CFTR genotypes, compound heterozygosity was the rule, and the most frequent CBAVD combinations were ΔF508/5T (35%), ΔF508/other mutation (30%, including ΔF508/R117H-7T: 5,6%), and 5T/other mutation (17%). No combination of two severe mutations was found in CBAVD (0%); by contrast with the CF population, 88% of genotypes identified in CBAVD comprised a severe mutation in trans to a mild mutation, and 12% consisted of 2 mild mutations. A total of 22 genotypes were shared by both CF and CBAVD. The role of the 5T allele as a splicing variant with variable, incomplete disease penetrance in CBAVD is reviewed. Other haplotype backgrounds, such as the TG12 sequence and the M470V polymorphism, may influence CFTR splicing and/or function. This study confirms the high molecular heterogeneity of CFTR mutations in CBAVD and emphasizes the importance of extensive CFTR analysis in these patients. Longterm follow-up studies of CBAVD patients are necessary in order to predict the phenotypic consequences of numerous CFTR mutational genotypes.     

Functional Expression of p64, an Intracellular Chloride Channel Protein

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity. Received: 17 November 1997/Revised: 9 February 1998     

Chemical synthesis of phosphoseryl-phosphoserine, a partial analogue of human salivary statherin, a protein inhibitor of calcium phosphate precipitation in human saliva

Human salivary secretions are supersaturated with respect to basic calcium phosphates but spontaneous precipitation of these salts from saliva, or surface-induced precipitation of calcium phosphates onto dental enamel, does not normally occur. This unexpected stability has been attributed to the inhibitory activities of two kinds of salivary phosphoproteins: statherin and the acidic, proline-rich phosphoproteins (PRP). Investigation of the structure-function relationships of statherin, the most potent inhibitor of primary (spontaneous) and secondary (seeded) precipitation of calcium phosphate salts in human saliva has been limited to studies of peptide segments obtained from the native peptide by specific proteolysis. Solid phase peptide synthesis (SPPS) is a useful and potentially more flexible alternative. Phosphoserine residues (positions 2 & 3) play critically important roles in the precipitation-inhibition activities of statherin, but SPP synthesis of these phosphorylated peptides is precluded because of the instability of phosphoserine residues in the presence of HF. Thus, this peptide was synthesized by solution-phase methods. The dipeptide possessed substantial inhibitory activity in assays for inhibition of both primary and secondary precipitation of calcium phosphate salts, but was not as active as either N-terminal tryptic hexapeptide of statherin or intact statherin. Syntheses of other model phosphorylated peptides are underway to expand the structure-function relationships.     

Could the elongate yellow-orange nostrils of Anguilla bicolor McClelland, 1844 function as fishing lures?

The shortfin eel Anguilla bicolor has elongate, yellow nostrils tipped with orange that protrude forward above the mouth. They are a striking, highly visible feature and it is hypothesised that they function as lures to attract prey, analogous to the illicium and esca of anglerfishes and frogfishes and the lingual appendage of the alligator snapping turtle. Another possible function is as an intraspecific signalling device. The first hypothesis is favoured here.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.